The osmolality of NEUROBASAL -A was demonstrated to be optimal for postnatal and adult CNS neurons.

NEUROBASAL when supplemented with N23, B27 or serum is effective for the growth of tumor cell lines of neuronal origin.

NEUROBASAL -A Cat. No. 12349 - Specific use for receptor studies such as estrogenic receptors, downstream protein purification studies or other processes where the presence of Phenol Red is undesirable

Background

NEUROBASAL with B27 (Cat. No. 17504) has shown excellent long-term viability of rat embryonic hippocampal neurons even after four (4) weeks in culture with greater than 90% viability for cells plated at 640/mm2 and
greater than 50% viability for cells plated at 160/mm2. Glial cell growth at five (5) days is reduced to less than 0.5% for a nearly pure neuronal population
1 .

The growth of adult CNS neurons requires gentle separation of their numerous connections, a density gradient for the separation of oligodendrocytes and enrichment of neurons, an adequate substrate for attachment and a dedicated medium for growth. Brewer1 has shown that NEUROBASAL -A supplemented with B27 and adequate isolation methods permit the isolation of spherical remnants of hippocampal neurons from any age rat and promote the regeneration of axon and dendrite-like processes.

Neurobasal and Neurobasal -A must be supplemented with N2 or B27 and 0.5 mM L-glutamine. For the initial plating of embryonic primary hippocampal neurons, it is suggested that 25 μM (3.7 μg/mL) glutamate be added to the NEUROBASAL medium. Both media support the growth of nearly pure populations of neural cells without the need of an astrocyte feeder layer. Both media, when supplemented with B27, contain antioxidants to reduce reactive oxygen damage and they do not contain the excitatory amino acids, glutamate and aspartate, making it amenable to the study of these neurotransmitters.

c. One hour after plating and incubation (ambient oxygen with 5 % CO2 is acceptable but 9 % oxygen with 5 % CO2 is preferable), the coverslip is quickly picked up, allowed to drain and transferred to 0.4 mL NEUROBASAL -A/B27 in a 24-well plate at 37° C. For postnatal neurons, the medium is aspirated, the coverslips rinsed once with warm NEUROBASAL -A and the cells re-fed with NEUROBASAL -A supplemented with B27, 0.5 mM L-glutamine, 1% penicillin-streptomycin (Cat. No. 15070) and 5 ng/mL β-FGF (Cat. No. 13256).

d. When culturing the cells for longer than 4 days, one-half of the medium is removed on day 3 or 4 and replaced with an equal volume of medium now containing 10 ng/mL β-FGF (postnatal neurons). Subsequent medium changes for prenatal neurons (4 days) should be made with NEUROBASAL without glutamate, to reduce glutamate toxicity in the culture. With neuroblastomas, the glutamate should be included in the medium for both plating and subsequent media changes.

Improved long-term survival of hippocampal neurons may be obtained by the addition of 2-mercaptoethanol (Cat. No. 21985) at 25 μM
4.

Quality Control Testing

NEUROBASAL - formulations are tested for pH, osmolality, endotoxin and the absence of bacterial and fungal contamination. For growth promotion and absence of toxicity, the medium is supplemented with B27 and tested in a growth assay utilizing a B104 (neuroblastoma) cell line